Hemoglobin-based oxygen carriers (“HBOC”) have long been associated with vasoconstriction that has been attributed to nitric oxide (NO) scavenging by heme. Oxygen carriers that are useful as oxygen therapeutics (sometimes referred to as “oxygen-carrying plasma expanders”), such as stabilized hemoglobin (Hb), have been shown to have limited efficacy because they scavenge nitric oxide, causing vasoconstriction and hypertension. The propensity of these oxygen carrying solutions to cause vasoconstriction can manifest as hypertension in animals and man. Although the mechanisms underlying the vasoconstrictive effects of HBOCs are not well understood, it has been suggested that the heme iron may combine rapidly and irreversibly with endogenous NO, a powerful vasodilator, thereby causing vasoconstriction.
In part because of these vasoconstrictive effects, no oxygen carrier to date has been entirely successful as an oxygen therapeutic agent (OTA), although products comprising modified cell-free Hb have been the most promising. Human Hb cross-linked between α-chains with bis-dibromosalicyl-fumarate (ααHb) was developed by the U.S. Army as a model red cell substitute, but was abandoned after it exhibited severe increases in pulmonary and systemic vascular resistance (Hess, J. et al., 1991, Blood 78:356A). A commercial version of this product was also abandoned after a disappointing Phase III clinical trial (Winslow, R. M., 2000, Vox Sang 79:1-20).
Two molecular approaches have been advanced in attempting to overcome the NO binding activity of Hb. The first approach used site-directed mutagenesis of the distal heme pocket in an attempt to create a recombinant hemoglobin with reduced NO-binding affinity (Eich, R. F. et al., 1996, Biochem. 35:6976-83). The second approach used a chemical modification approach wherein the size of the Hb was enhanced through oligomerization in an attempt to reduce or possibly completely inhibit the extravasation of Hb from the vascular space into the interstitial space (Hess, J. R. et al., 1978, J. Appl. Physiol. 74:1769-78; Muldoon, S. M. et al., 1996, J. Lab. Clin. Med. 128:579-83; Macdonald, V. W. et al., 1994, Biotechnology 22:565-75; Furchgott, R., 1984, Ann. Rev. Pharmacol. 24:175-97; and Kilbourne, R. et al., 1994, Biochem. Biophys. Res. Commun. 199:155-62).
In fact, recombinant Hbs with reduced association binding rates for NO have been produced that are less hypertensive in top-load rat experiments (Doherty, D. H. et al. 1998, Nature Biotechnology 16:672-676 and Lemon, D. D. et al. 1996, Biotech 24:378). However, studies suggest that NO binding may not be the only explanation for the vasoactivity of Hb. It has been found that certain large Hb molecules, such as those modified with polyethylene glycol (PEG), were virtually free of vasoconstriction, even though their NO association rates were identical to those of the severely hypertensive ααHb (Rohlfs, R. J. et al. 1998, J Biol. Chem. 273:12128-12134). Furthermore, it was found that PEG-Hb was extraordinarily effective in preventing the consequences of hemorrhage when given as an exchange transfusion prior to hemorrhage (Winslow, R. M. et al. 1998, J. Appl. Physiol. 85:993-1003).
The conjugation of PEG to Hb reduces its antigenicity and extends its circulation half-life. However, the PEG conjugation reaction has been reported to result in dissociation of Hb tetramers into αβ-dimer subunits causing gross hemoglobinuria in exchange-transfused rats receiving PEG-conjugates of Hb monomeric units below 40,000 Daltons (“Da”) (Iwashita and Ajisaka Organ-Directed Toxicity: Chem. Indicies Mech., Proc. Symp., Brown et al. 1981, Eds. Pergamon, Oxford, England pgs 97-101). A polyalkylene oxide (“PAO”) conjugated Hb having a molecular weight greater than 84,000 Daltons was prepared by Enzon, Inc. (U.S. Pat. No. 5,650,388) that carried about 10 copies of PEG-5,000 chains linked to Hb at its α and ε-amino groups. This degree of substitution was described as avoiding clinically significant nephrotoxicity associated with hemoglobinuria in mammals. However, the conjugation reaction resulted in a heterogeneous conjugate population and contained other undesirable reactants that had to be removed by column chromatography.
PEG conjugation is typically carried out through the reaction of an activated PEG moiety with a functional group on the surface of biomolecules. The most common functional groups are the amino groups of lysine, imidazole groups of histidine residues, and the N-terminus of proteins; thiol groups of cysteine residues; and the hydroxyl groups of serine, threonine and tyrosine residues and the C-terminus of the protein. PEG is usually activated by converting the hydroxyl terminus to a reactive moiety capable of reacting with these functional groups in a mild aqueous environment. One of the most common monofunctional PEGs used for conjugation of therapeutic biopharmaceuticals is methoxy-PEG (“mPEG-OH”), which has only one functional group (i.e. hydroxyl), thus minimizing cross-linking and aggregation problems that are associated with bifunctional PEG. However, mPEG-OH is often contaminated with high molecular weight bifunctional PEG (i.e. “PEG diol”), which can range as high as 10 to 15% (Dust J. M. et al. 1990, Macromolecule 23:3742-3746) due to its production process. This bifunctional PEG diol has roughly twice the size of the desired monofunctional PEG. The contamination problem is further aggravated as the molecular weight of PEG increases. The purity of mPEG-OH is especially critical for the production of PEGylated biotherapeutics, because the FDA requires a high level of reproducibility in the production processes and quality of the final drug product.
Conjugation of Hb to PAOs has been performed in both the oxygenated and deoxygenated states. U.S. Pat. No. 6,844,317 describes conjugating Hb in the oxygenated, or “R” state by equilibrating Hb with the atmosphere prior to conjugation to enhance the oxygen affinity of the resultant PEG-Hb conjugate. Others describe a deoxygenation step prior to conjugation to diminish the oxygen affinity and increase structural stability, enabling the Hb to withstand the physical stresses of chemical modification, diafiltration and/or sterile filtration and pasteurization (U.S. Pat. No. 5,234,903). For intramolecular cross-linking of Hb, it is suggested that deoxygenating Hb prior to modification may be required to expose lysine 99 of the α-chain to the cross-linking reagent (U.S. Pat. No. 5,234,903).
The kinetics of Hb thiolation with 2-iminothiolane prior to conjugation with PEG was investigated by Acharya et al. (U.S. Pat. No. 7,501,499). It was observed that increasing the concentration of iminothiolane from 10-fold, which introduced an average of five extrinsic thiols per tetramer, to 30-fold nearly doubled the number of extrinsic thiols on Hb. However, the size enhancement seen after PEG conjugation was only marginal, even with double the number of thiols. This suggested that the conjugation reaction in the presence of 20-fold molar excess of maleimidyl PEG-5000 covered the surface of the Hb with less reactive thiols, resulting in steric interference that resisted further modification of Hb with more reactive thiols. Consequently, to achieve the desired degree of conjugation of modified Hb (i.e. 6±1 PEG per Hb molecule), Acharya et al. thiolated Hb with an 8-15 fold molar excess of iminothiolane, and then reacted the thiolated Hb with a 16-30 fold molar excess of maleimidyl PEG-5000. However, these high molar excess reactant concentrations in large-scale production significantly increase the cost for preparing the HBOC and increase the heterogeneity of the final product. Moreover, such high molar excess of the maleimidyl PEG-5000 also results in a more heterogeneous product with the production of a greater number of unwanted side reactants.
In previous studies, it was observed that the molecular size of surface modified hemoglobin has to be large enough to avoid being cleared by the kidneys and to achieve the desired circulation half-life. Blumenstein, J. et al., determined that this could be achieved at, or above, a molecular weight of 84,000 Daltons (“Da”) (“Blood Substitutes and Plasma Expanders,” Alan R. Liss, editors, New York, N.Y., pages 205-212 (1978)). In that study, the authors conjugated dextran of varying molecular weight to Hb. They reported that a conjugate of Hb (with a molecular weight of 64,000 Da) and dextran (having a molecular weight of 20,000 Da) “was cleared slowly from the circulation and negligibly through the kidneys.” Further, it was observed that increasing the molecular weight above 84,000 Da did not significantly alter these clearance curves. Intramolecular cross-linking chemically binds together subunits of the tetrameric hemoglobin unit to prevent the formation of dimers which are prematurely excreted by the kidney. (See, e.g., U.S. Pat. No. 5,296,465.
Nitrite reacts with oxyhemoglobin to form methemoglobin, and reacts with deoxyhemoglobin to form methemoglobin and nitric oxide. The vasodilatory effect of nitrite differs from that of traditional NO donors in the presence of hemoglobin and can in part be explained by the nitrite reductase activity of hemoglobin. See Crawford et al. 2006 Blood 107:566-574; Huang et al. 2005 J Biol Chem 280:31126-31131; Huang et al. 2005 J Clin Invest 115:2099-2107. Studies have shown that nitrite is converted to NO only through reaction with deoxy hemes with the hemoglobin tetramer (Cosby, K. et al. 2003, Nat. Med. 9:1498), and further, that faster reduction of nitrite occurs where the protein heme is in the relaxed or R-state conformation. It is believed that this nitrite reductase activity of hemoglobin is under allosteric control and produces NO at a maximal rate when deoxygenated hemes are in an R-state conformation. R-state stabilizing effects can occur, for example, through modifications at βCys93 sites, such as maleimide PEG conjugation results in increased nitrite reductase activity. Further, it has been shown that while cell-free Hbs caused vasoconstriction and reduced perfusion, MalPEG-Hbs maintained blood flow and microvascular perfusion pressure, which is thought to be related to the lack of vasoconstriction (Tsai, A. G. et al. 2006, Blood 108:3603). Other studies also suggest that the modification of cell-free hemoglobin derivatives with multiple chains of PEG may suppress vasoactivity. Experiments utilizing R-State stabilized Hbs with five to six PEG chains demonstrated 10-fold faster nitrite reductase activity as compared to native Hb (Lui, F. E. et al. 2008, Biochemistry 47(40), 10773-10780). However, it was concluded that any further PEG conjugation at accessible lysine residues did not contribute to increased nitrite reductase activity.
Consequently, there is a need for a method of delivering oxygen, carbon monoxide, nitric oxide, or mixtures thereof to tissue and reducing nitrite to nitric oxide at an enhanced rate in the microvasculature through the use of a high oxygen affinity hemoglobin having increased nitrite reductase properties as compared to existing Hbs.